JP2018515089A - Penicillin G acylase - Google Patents

Abstract

The present invention relates to engineered penicillin G acylase (PGA) enzymes with improved properties, polynucleotides encoding such enzymes, compositions comprising the enzymes, and methods of using the enzymes. The present invention is an engineered penicillin G acylase capable of removing the A1 / B1 / B29 triphenylacetic acid protecting group from insulin to produce free insulin, comprising SEQ ID NOs: 2, 4, 6, 8, 10 And / or 12 and at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% A penicillin G acylase that is about 96%, about 97%, about 98%, about 99%, or more identical.

Description

  This application is a priority over US Provisional Patent Application No. 62 / 158,118, filed May 7, 2015, which is hereby incorporated by reference in its entirety for all purposes. Insist on the right.

Reference to Sequence Listing, Table or Computer Program File name CX2-149WO1_ST25.E in computer readable form (CRF) via EFS-Web under US Patent Law Enforcement Regulations §1.821. The sequence listing submitted at the same time as this application as txt is incorporated herein by reference. An electronic copy of the sequence listing was created on April 28, 2016, and the file size is 88 kilobytes.

  The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.

  Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzes the cleavage of the amide bond on the penicillin G (benzylpenicillin) side chain. This enzyme is used commercially in the production of 6-amino-penicillanic acid (6-APA) and phenyl-acetic acid (PAA). 6-APA is an important compound in the industrial production of semisynthetic β-lactam antibiotics such as amoxicillin, ampicillin and cephalexin. Naturally occurring PGA enzymes exhibit instability in commercial processes, which necessitates immobilization on a solid substrate for commercial application. PGA is covalently bound to various supports, and PGA immobilization systems have been reported as useful tools for synthesizing pure optical isomers. However, binding to a solid surface impairs enzyme properties, such as reduced activity and / or selectivity, and limits access to solutes. Furthermore, binding to a solid substrate allows the enzyme to be captured and reused in further processing cycles, but the stability of the enzyme is such that such applications can be limited. The enzymatic catalysis by PGA from penicillin G to 6-APA is regiospecific (does not cleave the lactamamide bond) and stereospecific. The production of 6-APA probably constitutes the greatest use of enzymatic catalysis in the production of pharmaceuticals. The enzymatic activity of PGA is related to the phenacetyl moiety and allows for stereospecific hydrolysis of a wide variety of phenacetyl derivatives of primary amines as well as alcohols.

  The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.

  The present invention is an engineered penicillin G acylase capable of removing the A1 / B1 / B29 triphenylacetic acid protecting group from insulin to produce free insulin, comprising SEQ ID NOs: 2, 4, 6, 8, 10 And / or 12 and at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% A penicillin G acylase that is about 96%, about 97%, about 98%, about 99%, or more identical. In some embodiments, the invention is an engineered penicillin G acylase that can remove A1 / B1 / B29 triphenylacetic acid protecting groups from insulin to produce free insulin, comprising SEQ ID NO: 2, 4, 6, 8, 10, and / or 12 and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Penicillin G acylases that are 97%, 98%, 99%, or more identical are provided. In some further embodiments, the invention is an engineered penicillin G acylase capable of removing the A1 / B1 / B29 triphenylacetic acid protecting group from insulin to produce free insulin, comprising SEQ ID NO: 2 Penicillin G acylases comprising 4, 6, 8, 10, and / or 12 are provided. In some further embodiments, the penicillin G acylase comprises at least one mutation provided in Table 5.1, Table 6.2, and / or Table 6.3.

  The present invention provides at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 90% to a sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11. Encoded by a polynucleotide sequence having sequence identity of 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more Penicillin G acylase is also provided.

  In some embodiments, the penicillin G acylase encoded by the polynucleotide sequence is at least 85%, 86%, 87%, 88 relative to a sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11. %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity. In some embodiments, the penicillin G acylase is encoded by a polynucleotide sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11. The present invention also provides vectors comprising the polynucleotide sequences presented herein (eg, SEQ ID NOs: 3, 5, 7, 9, and / or 11). The present invention also provides host cells comprising the vectors presented herein (eg, vectors comprising the polynucleotide sequences of SEQ ID NOs: 3, 5, 7, 9, and / or 11).

  The present invention is a method for producing free insulin comprising i) at least one engineered penicillin G acylase as presented herein and an A1 / B1 / B29 triphenylacetic acid protecting group And ii) the engineered penicillin G acylase is subjected to removal of the A1 / B1 / B29 triphenylacetic acid protecting group and free insulin is produced under conditions where the engineered penicillin G acylase is A1 / B1. Also provided is a method comprising exposing to an insulin comprising a / B29 triphenylacetic acid protecting group. In some embodiments of the method, the penicillin G acylase is at least about 85%, about 86%, about 87%, about 88%, about 89 with SEQ ID NOs: 2, 4, 6, 8, 10, and / or 12. %, About 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than the same . In some embodiments of the method, the penicillin G acylase is at least 85%, 86%, 87%, 88%, 89%, 90%, SEQ ID NO: 2, 4, 6, 8, 10, and / or 12. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical. In some further embodiments of the method, the penicillin G acylase comprises SEQ ID NOs: 2, 4, 6, 8, 10, and / or 12. In some embodiments, the engineered penicillin G acylase is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% Produces more than 99% or more free insulin. The present invention also provides compositions comprising free insulin produced according to the method (s) presented herein.

FIG. 1 provides a graph showing the substrate inhibitory activity observed with variant 1.

FIG. 2 provides a graph showing the amount of free insulin produced using the seven variants PGA.

FIG. 3 provides a graph showing the percent yield of free insulin produced using the three variants PGA.

FIG. 4 provides a graph showing the percent yield of free insulin produced using the three variants PGA in the presence of DMSO in the reaction.

  The present invention relates to an engineered penicillin capable of cleaving penicillin into phenylacetic acid and 6-aminopenicillanic acid (6-APA), an important intermediate in the synthesis of a wide variety of β-lactam antibiotics. G acylase (PGA) is provided. In particular, the present invention provides engineered PGAs that can remove A1 / B1 / B29 triphenylacetic acid protecting groups to release free insulin.

  In general, naturally occurring PGA is a heterodimeric enzyme composed of an α-subunit and a β-subunit. Wild-type PGA is naturally synthesized as a pre-pro-PGA polypeptide containing an N-terminal signal peptide that mediates translocation to the periplasm and a linker region that connects the C-terminus of the α subunit and the N-terminus of the β subunit. Is done. Protein processing results in the mature heterodimeric enzyme. The intermolecular linker region also functions in promoting proper folding of the enzyme. The PGAs described herein are based on PGAs derived from Kluyvera citrophila in which various modifications have been introduced to produce improved enzyme properties as described in detail below.

  In the context of the description presented herein, the use of the singular includes the plural (and vice versa) unless specifically stated otherwise. For example, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are interchangeable. And not limiting. Where the term “comprising” is used in the description of the various embodiments, in some specific examples, embodiments use the terms “consisting essentially of” or “consisting of” instead. It should be further understood that one skilled in the art would understand that

  Both the foregoing general description, including the figures, and the following detailed description are for purposes of illustration and description only and are not intended to limit the present disclosure. Further, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definitions As used herein, the following terms shall have the following meanings:

  For purposes of this disclosure, scientific and technical terms used in the description herein have the meaning commonly understood by one of ordinary skill in the art unless otherwise defined. Accordingly, the following terms shall have the following meanings: All patents and publications referred to herein are expressly incorporated by reference, including all sequences disclosed in such patents and publications. Unless otherwise specified, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology, and related fields, as known to those skilled in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Indeed, the particular methodology, protocols, and reagents described herein may vary depending on the context in which they are used, and the invention is not intended to be limited thereto. The titles presented herein are not intended to limit various aspects or embodiments of the invention.

  Nevertheless, in order to facilitate understanding of the present invention, a number of terms are defined below. Numerical ranges include numerical values that define the range. Accordingly, any numerical range disclosed herein is intended to encompass any narrow numerical range falling within such broad numerical range, where such narrow numerical range is all expressly recited herein. It is intended to be included as well. Any maximum (or minimum) numerical limit disclosed herein shall be construed herein as any lower (or higher) numerical limit, and such lower (or higher) numerical limits are explicitly defined herein. It is intended to encompass the same as described.

  As used herein, the term “comprising” and its congeners are used in their generic sense (ie, equivalent to the term “including” and its corresponding congeners). Is).

  As used herein and in the appended claims, the singular terms “a”, “an”, and “the” are not expressly specified by context. Unless stated, it includes a reference to the plural. Thus, for example, reference to (a) “host cell” includes a plurality of such host cells.

  Unless otherwise specified, each nucleic acid is written left to right in a 5 'to 3' direction, and the amino acid sequence is written left to right in an amino to carboxy direction, respectively.

  The titles presented herein are not intended to limit the various aspects or embodiments of the present invention that can be known by reference to the entire specification. Accordingly, the terms defined below are defined in more detail by reference to the entire specification.

  As used herein, “protein”, “polypeptide” and “peptide” as used herein are length or post-translational modifications (eg, glycosylation, phosphorylation, lipidation, myristylation, ubiquitin Used interchangeably to denote a polymer of at least 2 amino acids covalently linked by an amide bond, regardless of D-amino acids and L-amino acids and mixtures of D-amino acids and L-amino acids are included in this definition.

  As used herein, “polynucleotide” and “nucleic acid” refer to two or more nucleosides covalently linked together. A polynucleotide can be composed entirely of ribonucleosides (ie, RNA), can be composed entirely of 2 ′ deoxyribonucleotides (ie, DNA), or can be a mixture of ribonucleosides and 2 ′ deoxyribonucleosides. . Nucleosides are generally linked together via standard phosphodiester linkages, but polynucleotides can contain one or more non-standard linkages. A polynucleotide can be single-stranded or double-stranded, or can comprise both single- and double-stranded regions. In addition, polynucleotides are generally composed of naturally occurring coding nucleobases (ie, adenine, guanine, uracil, thymine and cytosine), but one or more modified and / or synthetic nucleobases (eg, inosine, Xanthine, hypoxanthine, etc.). Preferably, such modified or synthetic nucleobase is a coding nucleobase.

As used herein, “hybridization stringency” relates to hybridization conditions, such as wash conditions, in the hybridization of nucleic acids. In general, hybridization reactions are performed under conditions of lower stringency, followed by higher stringency washes, albeit at various stringencies. The term “moderately stringent hybridization” means that the target DNA is about 60% identical to the target DNA, preferably about 75% identical, with greater than about 90% identity to the target polynucleotide. Refers to conditions that allow binding to a complementary nucleic acid having about 85% identity. Exemplary moderately stringent conditions are those equivalent to hybridization in 50% formamide, 5 × Denhardt's solution, 5 × SSPE, 0.2% SDS at 42 ° C. Washing in 0.2 × SSPE, 0.2% SDS follows at 0 ° C. “High stringency hybridization” generally refers to conditions at or below about 10 ° C. from the thermal melting temperature T _m determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65 ° C. (ie, hybrids of 65 If it is not stable in 0.018M NaCl at 0 ° C., it is not stable under high stringency conditions as intended herein). High stringency conditions include, for example, hybridization at 42 ° C. under conditions equivalent to 50% formamide, 5 × Denhardt's solution, 5 × SSPE, 0.2% SDS, followed by 65 ° C. with 0.1% XSSPE, and can be brought about by washing in 0.1% SDS. Another high stringency condition is to hybridize in 5 × SSC containing 0.1% (w: v) SDS at 65 ° C. and contain 0.1% SDS at 65 ° C. Hybridization under conditions equivalent to washing in 0.1 × SSC. Other high stringency hybridization conditions, as well as moderately stringent conditions, are known to those skilled in the art.

  As used herein, “coding sequence” refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

  As used herein, “codon optimized” means that the codons of a polynucleotide encoding a protein are those of a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. It means to change to the one that is used preferentially within. In some embodiments, a polynucleotide encoding a PGA enzyme can be codon optimized for optimal production from a host organism selected for expression. The genetic code is degenerate in that most amino acids are represented by several codons called “synonymous” codons or “synonymous” codons, but the codon usage by a particular organism (codon It is well known that usage) is non-random and biased to a specific codon triplet. This codon usage bias can be higher for a given gene, a gene of common function or ancestral origin, a highly expressed protein, and a low copy number protein, and an aggregated protein coding region of an organism's genome. In some embodiments, a polynucleotide encoding a PGA enzyme can be codon optimized for optimal production from a host organism selected for expression.

  As used herein, “preferred, optimal, high codon usage biased codon” refers to codons used more frequently in a protein coding region than other codons that encode the same amino acid. Point to. Preferred codons are one gene, a series of genes of common function or origin, codon usage in highly expressed genes, codon frequency in aggregated protein coding regions of the entire organism, aggregation of related organisms It can be determined with respect to the codon frequency in the protein coding region, or a combination thereof. Codons whose frequency increases with the level of gene expression is generally the optimal codon for expression. Various methods for determining codon frequency (eg, codon usage, relative synonymous codon usage) and codon preference in a particular organism, including multivariate analysis, eg, using cluster analysis or correspondence analysis, and Various methods are known for determining the effective number of codons used in a gene (eg GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, John Peden, University of Nottingham; McInerney, Bioinform., 14: 372-73, [1998]; Stenico et al., Nucleic Acids Res., 222: 437-46, [1994]; and Wright, Gene 87: 23-29, [1990]. ) For an expanding list of organisms, a codon usage frequency table is available (eg, Wada et al., Nucleic Acids Res., 20: 2111-2118, [1992]; Nakamura et al., Nucl. Acids Res., 28: 292, [2000]; see Duret et al., Supra; Henaut and Danchin, “Escherichia coli and Salmonella”, Neidhardt et al. (Ed.), ASM Press, Washington DC, [1996], 2047-2066. ) A data source for obtaining codon usage can rely on any available nucleotide sequence that can encode a protein. These data sets include the predicted coding region of a nucleic acid sequence that is actually known to encode the expressed protein (eg, complete protein coding sequence-CDS), expressed sequence tag (ESTS), or genomic sequence. (See, eg, Uberbacher, Meth. Enzymol., 266: 259-281, [1996]; Tiwari et al., Comput. Appl. Biosci., 13: 263-270, [1997]). .

  As used herein, a “control sequence” is defined herein to include all components necessary or advantageous for expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence can be native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.

  As used herein, “operably linked” is used herein to mean that the control sequence is suitably positioned at a position relative to the polynucleotide of interest (ie, functionally As a result, the regulatory sequence is defined as the configuration that directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

  As used herein, “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The control sequence may include a suitable promoter sequence. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. A promoter, including mutant promoters, shortened promoters and hybrid promoters, can be any nucleic acid sequence that exhibits transcriptional activity in the selected host cell and encodes an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. Can be obtained from the gene

  As used herein, “naturally occurring” or “wild type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism and can be isolated from a natural source and is intentionally modified by human manipulation. It is not an array.

  As used herein, “non-naturally occurring”, “recombinant” or “artificial”, when used in this disclosure with respect to (eg, a cell, nucleic acid or polypeptide), is modified in some manner. Or a material that corresponds to the natural or native form of the material and that does not exist in nature unless modified. In some embodiments, the material is the same as the naturally occurring material, but the material produced or produced from synthetic materials and / or by manipulation using recombinant techniques, or the natural form or native of that material Refers to a material corresponding to a particular form. Non-limiting examples include sets that express genes that are not found in native (non-recombinant) forms of cells, or that express native genes that are expressed at different levels in the absence of modification. Replacement cells can be mentioned.

As used herein, “percent sequence identity”, “percent identity” and “percent identity” refer to a comparison between polynucleotide sequences or between polypeptide sequences and optimal alignment over the comparison window. A portion of the polynucleotide or polypeptide sequence within the comparison window is compared to the reference sequence for optimal alignment of the two sequences, compared to the reference sequence in the optimal alignment of the two sequences (ie, Gap). Determine the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is present in both sequences, or where the nucleobase or amino acid residue is aligned with the gap, and the number of match positions is the comparison window The best alignment and percent sequence identity determination to calculate this percentage by dividing by the total number of positions within and multiplying the result by 100 to determine the percentage of sequence identity uses the BLAST and BLAST 2.0 algorithms (Eg, Altschul et al., J. Mol. Biol., 215: 403-410, [1990]; and Altschul et al., Nucl. Acids Res., 3389-3402, [1977]. checking). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information website.

  Briefly, the BLAST analysis, when aligned with words of the same length in the database sequence, has a short word length W in the query sequence that matches or satisfies some positive threshold score T. First identifying a high-scoring sequence pair (HSP). T is referred to as the neighborhood word score threshold (Altschul et al., Ibid). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hit is then expanded in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, parameters M (matching residue pair reward score; always> 0) and N (mismatched residue penalty score; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Whether the cumulative alignment score is reduced by an amount X from its maximum achieved value; by accumulating one or more negative score residue alignments, the cumulative score is less than zero; or when the end of any sequence is reached , Stop expanding word hits in each direction. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default 11 word lengths (W), 10 expected values (E), M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (eg, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 [1989]).

  Many other algorithms are available and are known in the art that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison is performed using any suitable method known in the art (eg, Smith and Waterman, Adv. Appl. Math., 2: 482, [1981] By the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, [1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85 Volume: 2444, [1988] by similarity studies; and / or by computer-controlled execution of these algorithms [GAP, BESTFIT, FASTA, and TFASTA in GCG Wisconsin Software Package]), or the technology By visual inspection using methods generally known in the field It kills. In addition, the BESTFIT or GAP program in the GCG Wisconsin Software Package can be used to determine sequence alignment and percent sequence identity using the default parameters provided (Accelrys, Madison WI).

  As used herein, “substantial identity” is when compared to a reference sequence over a comparison window of at least 20 residue positions, and often over a window of at least 30-50 residues. Refers to a polynucleotide or polypeptide sequence having at least 80 percent sequence identity, at least 85 percent identity, and 89-95 percent sequence identity, more usually at least 99 percent sequence identity. The percentage of sex is calculated by comparing the reference sequence to a sequence containing deletions or additions that total no more than 20 percent of the reference sequence over the window of comparison. In certain embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences are optimally aligned, such as by program GAP or BESTFIT, using default gap weights. Means sharing at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or greater (eg, 99 percent sequence identity) To do. In some preferred embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

  As used herein, “reference sequence” refers to a defined sequence relative to another sequence to be compared. A reference sequence can be a subset of a larger sequence, eg, a segment of a full-length gene or polypeptide sequence. Generally, the reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of a nucleic acid or polypeptide. Each of the two polynucleotides or polypeptides can include (1) a sequence that is similar between the two sequences (ie, part of the complete sequence), and (2) a sequence that is very different between the two sequences. In general, two (or more) polynucleotides or polypeptides by comparing the sequence of two polynucleotides over a comparison window to identify and compare the sequence similarity of local regions A sequence comparison between is performed. The term “reference sequence” is not limited to wild-type sequences, but can include engineered or altered sequences. For example, in some embodiments, a “reference sequence” can be a pre-engineered or altered amino acid sequence.

  As used herein, a “comparison window” refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein the sequence is a reference sequence of at least 20 consecutive nucleotides or amino acids. The portion of the sequence in the comparison window can contain no more than 20 percent additions or deletions when compared to a reference sequence (not including additions or deletions) with respect to optimal alignment of the two sequences. That is, a gap) may be included. The comparison window may be consecutive residues longer than 20, optionally including 30, 40, 50, 100 or more long windows.

  As used herein, “corresponds to”, “with reference to” and “compared with” when used in the context of numbering a given amino acid or polynucleotide sequence Refers to the numbering of residues of a particular reference sequence when the amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is specified relative to a reference sequence, not by the actual numerical position of the residue within a given amino acid sequence or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an artificial PGA, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, there are gaps, but the numbering of the residues in a given amino acid sequence or polynucleotide sequence is performed with respect to the reference sequence to which this sequence is aligned. As used herein, references to residue positions such as “Xn” further described below are to be construed as references to “residues corresponding to” unless otherwise indicated. Should be. Thus, for example, “X94” refers to any amino acid at position 94 of a polypeptide sequence.

  As used herein, “improved enzyme properties” refers to PGAs that exhibit some improvement in enzyme properties when compared to a reference PGA. For the engineered PGA polypeptides described herein, the comparison is generally made to the wild type PGA enzyme, but in some embodiments, the reference PGA is another improved engineered PGA. It can be. Enzymatic properties that are desired to be improved include, but are not limited to, enzyme activity (which can be expressed in terms of conversion rate of the substrate at a specific reaction time using a specific amount of PGA), thermal stability, solvent stability Sex, pH activity profile, cofactor requirements, refractory to inhibitors (eg, product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

As used herein, “increased enzyme activity” refers to an improved property of an engineered PGA polypeptide that is specific activity (eg, produced product) compared to a reference PGA enzyme. Product / time / protein weight) or conversion of substrate to product (eg, conversion of starting substrate to product over a specific period of time using a specific amount of PGA) Can be represented by an increase. Exemplary methods for determining enzyme activity are provided in the examples. Any property related to enzyme activity, including the classical enzyme properties of K _m , V _max or k _cat , can be affected and the change can lead to an increase in enzyme activity. The improvement in enzyme activity is from about 1.5 times the enzyme activity of the corresponding wild-type PGA enzyme, 2 to 5 times over the naturally occurring PGA from which the PGA polypeptide is derived, or another engineered PGA. It can be up to 10 times, 20 times, 25 times, 50 times, 75 times, 100 times or even more enzyme activity. In certain embodiments, the engineered PGA enzyme exhibits improved enzyme activity in the range of 1.5-50 times, 1.5-100 times that of the parent PGA enzyme. Those skilled in the art will appreciate that the activity of either enzyme is diffusion limited and therefore the catalytic turnover rate cannot exceed the diffusion rate of the substrate containing any necessary cofactors. . The theoretical maximum of diffusion limited or k _cat / K _m is generally about 10 ⁸ to 10 ⁹ (M ⁻¹ s ⁻¹ ). Thus, any improvement in the enzymatic activity of PGA has an upper limit related to the diffusion rate of the substrate affected by the PGA enzyme. PGA activity can be measured by any one of the standard assays used to measure the release of phenylacetic acid upon cleavage of penicillin G, such as by titration (eg, by titration). Simons and Gibson, Biotechnol. Tech., 13: 365-367, [1999]). In some embodiments, PGA activity is determined by the cleavage product 5-amino-2-nitro-benzoic acid, which is spectrophotometrically detectable (λmax = 405 nm) 6-nitrophenylacetamide benzoic acid (NIPAB). It can be measured by using. The comparison of enzyme activity is performed using a defined preparation of the enzyme, a defined assay under certain conditions, and one or more defined substrates, as described in further detail herein. In general, when lysates are compared, the same expression system and the same host cell are used to minimize variation in the amount of enzyme produced by the host cell and present in the lysate, The number of cells and the amount of protein assayed are determined.

As used herein, “increased enzyme activity” and “increased activity” refer to improved properties of the engineered enzyme compared to the reference enzyme described herein. Specific activity (eg, product produced / time / weight of protein) or conversion rate of substrate to product (eg, starting amount of substrate using a specific amount of PGA for a specific period of time (Conversion rate to product). Any property related to enzyme activity, including the classical enzyme properties of K _m , V _max or k _cat , can be affected and the change can lead to an increase in enzyme activity. In some embodiments, the PGA enzymes presented herein release insulin by removing a triphenylacetic acid protecting group from a particular residue of insulin. The comparison of enzyme activity is performed using a defined preparation of the enzyme, a defined assay under certain conditions, and one or more defined substrates, as described in further detail herein. In general, when enzymes in cell lysates are compared, the same expression system and the same host cell are used to minimize variation in the amount of enzyme produced by the host cell and present in the lysate. In addition, the number of cells and the amount of protein assayed are determined.

  As used herein, “conversion” refers to the enzymatic conversion of a substrate to the corresponding product.

  As used herein, “conversion” refers to the percentage of substrate that is converted to product within a period of time under certain conditions. Thus, for example, the “enzymatic activity” or “activity” of a PGA polypeptide can be expressed as the “conversion rate” of a substrate to a product.

  As used herein, “chemoselectivity” refers to the formation of one product preferentially over another in a chemical or enzymatic reaction.

  As used herein, “thermostable” and “thermal stable” are a series of temperature conditions (eg, 40-80) over a period of time (eg, 0.5-24 hours). When exposed to a certain level of residual activity (eg, greater than 60% to 80%) after exposure to elevated temperatures. %) Are used interchangeably.

  As used herein, “solvent stable” refers to various concentrations (eg, 5-99%) of solvents (eg, isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, acetic acid). Butyl, methyl tert-butyl ether, etc.) after exposure for a period of time (e.g. 0.5-24 hours) followed by similar activity (e.g. Refers to the ability of a polypeptide to maintain.

  As used herein, “pH stable” means after exposure to high or low pH (eg, 4.5-6 or 8-12) for a period of time (eg, 0.5-24 hours). , Refers to PGA polypeptides that maintain similar activity (eg, greater than 60% to 80%) compared to untreated enzyme.

  As used herein, “thermal and solvent stable” refers to a PGA polypeptide that is both thermostable and solvent stable.

  As used herein, “hydrophilic amino acid or residue” refers to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol. 179: 125-142, [ 1984]) refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than 0. The hydrophilic amino acid encoded by the gene includes L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln ( Q), L-Asp (D), L-Lys (K) and L-Arg (R).

  As used herein, “acidic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain that exhibits a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. . Acidic amino acids generally have negatively charged side chains due to loss of hydrogen ions at physiological pH. Acidic amino acids encoded by the gene include L-Glu (E) and L-Asp (D).

  As used herein, a “basic amino acid or residue” is a hydrophilic amino acid or residue having a side chain that exhibits a pK value greater than about 6 when the amino acid is included in a peptide or polypeptide. Point to. Basic amino acids generally have positively charged side chains due to association with hydronium ions at physiological pH. Examples of basic amino acids encoded by the gene include L-Arg (R) and L-Lys (K).

  As used herein, a “polar amino acid or residue” is uncharged at physiological pH, but the electron pair shared by two atoms is more closely held by one of the atoms It refers to a hydrophilic amino acid or residue having a side chain with at least one bond. The polar amino acids encoded by the gene include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).

  As used herein, “hydrophobic amino acids or residues” refers to the Eisenberg et al. Normalized consensus hydrophobicity scale (Eisenberg et al., J. Mol. Biol. 179: 125-142, [ 1984]) refers to an amino acid or residue having a side chain that exhibits a hydrophobicity greater than zero. Hydrophobic amino acids encoded by genes include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp ( W), L-Met (M), L-Ala (A) and L-Tyr (Y).

  As used herein, “aromatic amino acid or residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain comprising at least one aromatic or aromatic heterocycle. Aromatic amino acids encoded by the gene include L-Phe (F), L-Tyr (Y) and L-Trp (W). L-His (H) is sometimes classified as a basic residue based on the pKa of the nitrogen atom of the aromatic heterocycle, or an aromatic residue due to the side chain containing an aromatic heterocycle However, as used herein, histidine is classified as a hydrophilic residue or “constrained residue” (see below).

  As used herein, “constrained amino acid or residue” refers to an amino acid or residue having a constrained geometry. As used herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline also has a constrained geometric shape because it has a five-membered ring.

  As used herein, a “nonpolar amino acid or residue” is uncharged at physiological pH, and an electron pair shared by two atoms is generally held equally by each of the two atoms. Refers to a hydrophobic amino acid or residue having a side chain with a bond that is made (ie, the side chain is not polar). Nonpolar amino acids encoded by genes include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala ( A).

  As used herein, “aliphatic amino acid or residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Aliphatic amino acids encoded by the gene include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I).

  Cysteine (or “L-Cys” or “[C]”) is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl-containing or sulfhydryl-containing amino acids. Keep in mind. “Cysteine-like residues” include cysteine and other amino acids containing sulfhydryl moieties that can be used to form disulfide bridges. The ability of L-Cys (C) (and other amino acids with side chains containing -SH) to be present in the peptide in either reduced free -SH or oxidized disulfide bridged form is determined by the L-Cys Affects whether (C) contributes to the net hydrophobic or hydrophilic character of the peptide. L-Cys (C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), but for the purposes of this disclosure, L-Cys (C) It should be understood that they are categorized into their own groups.

  As used herein, a “small amino acid or residue” has a side chain composed of a total of three or less carbon and / or heteroatoms (except α-carbon and hydrogen). Refers to an amino acid or residue. Small amino acids or residues can be further categorized into aliphatic, nonpolar, polar or acidic small amino acids or residues according to the definition above. Small amino acids encoded by genes include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T ) And L-Asp (D).

  As used herein, “hydroxyl-containing amino acid or residue” refers to an amino acid containing a hydroxyl (—OH) moiety. Amino acids containing the hydroxyl encoded by the gene include L-Ser (S) L-Thr (T) and L-Tyr (Y).

  As used herein, “amino acid difference” and “residue difference” refer to the difference in amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as “Xn”, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, “residue difference at position X40 compared to SEQ ID NO: 2” refers to the difference in amino acid residue at the polypeptide position corresponding to position 40 of SEQ ID NO: 2. Thus, when the reference polypeptide of SEQ ID NO: 2 has histidine at position 40, the “residue difference at position X40 compared to SEQ ID NO: 2” is the histidine at the position of the polypeptide corresponding to position 40 of SEQ ID NO: 2. Refers to an amino acid substitution at any residue other than. In most examples herein, the specific amino acid residue difference at a position is “XnY” (where “Xn” designates the corresponding position as described above, and “Y” appears in the artificial polypeptide. As a single letter identifier (ie, a different residue than in the reference polypeptide). In some cases, the disclosure also provides the conventional notation “AnB” (A is a single letter identifier of a residue in the reference sequence, and “n” is the number of the residue position in the reference sequence. And B is a single letter identifier of a residue substitution in the sequence of the artificial polypeptide). In some cases, a polypeptide of the present disclosure may contain one or more amino acid residue differences compared to a reference sequence, which is at a particular position where there are residue differences compared to the reference sequence. Indicated by the list. In some embodiments, where more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a “/” (eg, X192A / G). The present disclosure encompasses engineered polypeptide sequences that include one or more amino acid differences that include either or both conservative amino acid substitutions and non-conservative amino acid substitutions. The amino acid sequence of certain recombinant carbonic anhydrase polypeptides included in the sequence listing of the present disclosure includes an initiating methionine (M) residue (ie, M represents residue position 1). However, this initiating methionine residue may be removed by biological processing mechanisms, such as in host cells or in vitro translation systems, to create mature proteins that lack the initiating methionine residue but otherwise retain the properties of the enzyme. Those skilled in the art will appreciate that they can be removed. Thus, the term “amino acid residue difference compared to SEQ ID NO: 2 at position Xn” as used herein refers to the “Xn” position or corresponding position in a reference sequence that is processed to lack the starting methionine. (For example, (X-1) position n).

As used herein, the term “conservative amino acid substitution” refers to the interchangeability of a residue with a residue having a similar side chain, and thus generally the amino acid in a polypeptide is the same or similar. Substituting with an amino acid within the defined amino acid class. By way of example but not limitation, in some embodiments, an amino acid having an aliphatic side chain is substituted with another aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, and an amino acid having a hydroxyl side chain is Another amino acid having a hydroxyl side chain, such as serine and threonine, is substituted, and an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, such as phenylalanine, tyrosine, tryptophan, and histidine. An amino acid having a basic side chain is replaced with another amino acid having a basic side chain, such as lysine and arginine, and an amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (for example, aspartic acid Or glutamic acid) and / or hydrophobic or Aqueous amino acids are respectively replaced with another hydrophobic or hydrophilic amino acids. Exemplary conservative substitutions are provided in Table 1.

  As used herein, “non-conservative substitution” refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions can use amino acids between defined groups rather than within defined groups: (a) the structure of the peptide backbone within the substitution (eg, glycine to proline) region, (b) charge or hydrophobicity Affects (c) side chain bulk. By way of example but not limitation, exemplary non-conservative substitutions include acidic amino acids substituted with basic or aliphatic amino acids, aromatic amino acids substituted with small amino acids, and hydrophilic amino acids substituted with hydrophobic amino acids. possible.

  As used herein, “deletion” refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion removes 1 amino acid or more, 2 amino acids or more, 5 amino acids or more, 10 amino acids or more, 15 amino acids or more, or 20 amino acids or more while retaining enzyme activity and / or retaining the improved properties of the artificial enzyme. However, it may include removal of up to 10% of the total number of amino acids comprising the polypeptide, or removal of up to 20% of the total number of amino acids comprising the reference enzyme. Deletions can be directed to the internal and / or terminal portions of the polypeptide. In various embodiments, the deletion can include continuous segments or can be discontinuous.

  As used herein, “insertion” refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved artificial PGA enzyme comprises one or more amino acid insertions into a naturally occurring PGA polypeptide, as well as one or more amino acid insertions into an artificial PGA polypeptide. The insertion can be at an internal portion of the polypeptide or can be to the carboxy terminus or amino terminus. As is known in the art, the insertions used herein include fusion proteins. In natural polypeptides, insertions can be contiguous amino acid segments or can be separated by one or more of the amino acids.

  The term “amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. The replacement set is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or There can be more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions present in any of the variant PGAs listed in the tables presented in the examples.

  As used herein, “fragment” refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion, but the remaining amino acid sequence being identical to the corresponding position in the sequence. Fragments can typically have about 80%, about 90%, about 95%, about 98% or about 99% of the full-length PGA polypeptide, eg, the polypeptide of SEQ ID NO: 2. In some embodiments, fragments are “biologically active” (ie, exhibit the same enzymatic activity as the full-length sequence).

  As used herein, an “isolated polypeptide” refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it, such as proteins, lipids and polynucleotides. The term encompasses polypeptides recovered or purified from their naturally occurring environment or expression system (eg, host cells or in vitro synthesis). Improved PGA enzymes can be present intracellularly, can be present in cell culture media, or can be prepared in various forms (eg, lysates or isolated preparations). Thus, in some embodiments, an artificial PGA polypeptide of the present disclosure can be an isolated polypeptide.

  As used herein, a “substantially pure polypeptide” is the predominant species in which the polypeptide species is present (ie, on a molar or weight basis, which is any other in the composition). Composition, which is more abundant than the individual polymer species), and generally a substantially purified composition when the target species comprises at least about 50 mole percent or about 50 weight percent of the polymer species present. It is a thing. In general, a substantially pure artificial PGA polypeptide composition is about 60 mol% or greater than or equal to about 70 mol% or greater than or equal to about 80 mol% of all macromolecular species present in the composition. Or about 90 mol% or more, or about 95 mol% or more, and about 98 mol% or more. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered high molecular species. In some embodiments, the isolated improved PGA polypeptide is a substantially pure polypeptide composition.

  As used herein, the term “heterologous” when used with reference to nucleic acids or polypeptides refers to sequences that are not normally expressed and secreted by an organism (eg, a wild-type organism). In some embodiments, the term is not found in the same relationship to each other as normally found in nature, or has been engineered so that the level of expression or other in the cell Includes sequences comprising two or more subsequences whose physical relationship with a nucleic acid or other molecule or structure is not normally found in nature. For example, a heterologous nucleic acid generally has two or more sequences derived from unrelated genes that are produced recombinantly and arranged in a manner not found in nature (eg, inserted into an expression cassette such as a vector). A nucleic acid open reading frame (ORF) of the present invention operably linked to a designated promoter sequence). In some embodiments, “heterologous polynucleotide” refers to any polynucleotide introduced into a host cell by laboratory techniques, removed from the host cell, subjected to laboratory manipulation, and then re-introduced into the host cell. Including the polynucleotide to be introduced.

  As used herein, “appropriate reaction conditions” are those conditions of a biocatalytic reaction solution that allows the PGA polypeptide of the present disclosure to release free insulin by removing the triphenylacetic acid protecting group ( For example, enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, co-solvent, etc.). Exemplary “suitable reaction conditions” are provided in this disclosure and are exemplified by the examples.

  As used herein, “load”, such as “compound load”, “enzyme load” or “cofactor load” refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

  As used herein, “substrate” in the context of a biocatalyst-mediated process refers to a compound or molecule on which the biocatalyst acts.

  As used herein, “product” in the context of a biocatalyst-mediated process refers to a compound or molecule resulting from the action of a biocatalyst.

  As used herein, “equilibrium” refers to a chemical or enzymatic reaction (eg, a stereoisomer interconversion, as determined by the forward and reverse rate constants of a chemical or enzymatic reaction (eg, Refers to a process that results in a steady state concentration of a chemical species in the interconversion of two species A and B).

  As used herein, “acylase” and “acyltransferase” are used interchangeably to refer to an enzyme capable of transferring an acyl group from a donor to an acceptor to form an ester or amide. The As a result of the acylase-mediated reverse reaction, the ester or amide is hydrolyzed.

As used herein, “penicillin G” and “benzylpenicillin” are (2S, 5R, 6R) -3,3-dimethyl-7-oxo-6- (2-phenylacetamido) -4-thia. -1-azabicyclo [3.2.0] also refers to known antibiotics as heptane-2-carboxylic acid _{(C 16 H 18 N 2 O} 4 S). This is mainly effective against gram positive organisms, but some gram negative organisms are also sensitive to it.

  As used herein, “penicillin G acylase” and “PGA” mediate cleavage of penicillin G (benzylpenicillin) to phenylacetic acid (PHA) and 6-aminopenicillanic acid (6-APA). Used interchangeably to refer to a competent enzyme. In some embodiments, the PGA activity is based on cleavage of the model substrate, eg, cleavage of 6-nitro-3- (phenylacetamido) benzoic acid to phenylacetic acid and 5-amino-2-nitro-benzoic acid. It can be. PGA can also perform a reverse reaction that transfers the acyl group of the acyl donor to the acyl acceptor. PGA, as used herein, includes naturally occurring (wild-type) PGA as well as non-naturally occurring PGA enzymes that include one or more engineered polypeptides made by human manipulation. . The wild type PGA gene is a heterodimer consisting of an α subunit (23.8 KDa) and a β subunit (62.2 KDa) linked by a 54 amino acid spacer region. Due to the presence of the spacer region, a self-processing step is required to form an active protein.

  As used herein, “acyl donor” refers to the portion of an acylase substrate that donates an acyl group to the acyl acceptor to form an ester or amide.

  As used herein, “acyl acceptor” refers to the portion of an acylase substrate that accepts the acyl group of an acyl donor to form an ester or amide.

  As used herein, “α chain sequence” refers to an amino acid sequence that corresponds to residues 27-235 of SEQ ID NO: 2 (eg, has at least 85% identity thereto). As used herein, a single chain polypeptide may include an “alpha chain sequence” and additional sequence (s).

  As used herein, “β chain sequence” refers to an amino acid sequence corresponding to (eg, having at least 85% identity to) residues 290-846 of SEQ ID NO: 2. As used herein, a single chain polypeptide may comprise a “β chain sequence” and additional sequence (s).

  As used herein, “derived from” as used in the context of an engineered PGA enzyme is the PGA enzyme from which the operation is based, and / or a gene encoding such a PGA enzyme. Is identified. For example, the engineered PGA enzyme of SEQ ID NO: 60 is It was obtained by artificially evolving genes encoding citrophila PGA α- and β-chain sequences over many generations. Thus, this engineered PGA enzyme is “derived from” the naturally occurring or wild type PGA of SEQ ID NO: 2.

  As used herein, “insulin” refers to a polypeptide hormone produced by pancreatic β cells in normal individuals. Insulin is necessary to regulate carbohydrate metabolism by lowering blood glucose levels. Diabetes is caused by a systemic deficiency of insulin. Insulin is composed of 51 amino acids and has a molecular weight of approximately 5800 daltons. Insulin is composed of two peptide chains (denoted by “A” and “B”) and contains one intrasubunit disulfide bond and two intersubunit disulfide bonds. The A chain is composed of 21 amino acids, and the B chain is composed of 30 amino acids. The two chains form a highly ordered structure with some α-helix regions in both the A and B chains. The isolated strand is inactive. In solution, insulin is monomeric, dimeric, or hexameric. Insulin is a hexamer in highly concentrated preparations used for subcutaneous injection, but becomes monomeric as it is diluted in body fluids. This definition refers to proinsulin and any purified isolated polypeptide having at least one of the major structural conformations and at least one biological property of naturally occurring insulin. It is intended to include. Furthermore, it is intended to encompass natural and synthetically produced insulin, including glycoforms, and analogs (eg, polypeptides having deletions, insertions, and / or substitutions).

  Insulin contains three nucleophilic amines that can potentially react with phenylacetic acid donors and can be deprotected by PGA. These residues include Lys at position 29 of the B chain (B29) and two N-terminal free amines, Gly at position 1 of the A chain (A1) and Phe at position 1 of the B chain (B1). The present invention provides triple protected insulin (phenylacetic acid chemically bound to the A1, B1 and B29 residues of human insulin). PGA has previously been reported to catalyze the hydrolysis of N-phenylacetic acid protected peptides and insulin with exclusive selectivity for phenylacetamide bonds, leaving the remaining peptide bonds of the protein intact. (Brtnik et al., Coll. Czech. Chem. Commun., 46 (8), 1983-1989, [1981]; and Wang et al., Biopolym., 25 (supplement), S109-S114, [ 1986]).

  As used herein, a “triphenylacetic acid protecting group” refers to three primary amines protected with phenylacyl groups at the B1, B29 and A1 positions as described herein. Refers to an insulin molecule having

Penicillin G acylase Penicillin acylase was prepared by Sakaguchi and Murao, Penicillium chrysogenum Wisc. First described from Q176 (Sakaguchi and Murao, J. Agr. Chem. Soc. Jpn., 23: 411, [1950]). Penicillin G acylase acts on the side chains of penicillin G, cephalosporin G, and related antibiotics, with phenylacetic acid as a common by-product, and is a β-lactam antibiotic intermediate, 6-amino It is a hydrolase that produces penicillanic acid and 7-aminodes-acetoxycephalosporanic acid. These antibiotic intermediates include potential components of semisynthetic antibiotics such as ampicillin, amoxicillin, cloxacillin, cephalexin, and cefatoxime.

As shown above, penicillin G acylase (PGA) is represented by Scheme 1:
As shown in FIG. 4, the hydrolysis of penicillin G having a conjugate base of structural formula (I) into 6-aminopenicillanic acid having a conjugate base of structural formula (II) and phenylacetic acid of structural formula (III). Characterized by the ability to catalyze cleavage by degradation.

Without being bound by theory, substrate specificity appears to be related to the recognition of hydrophobic phenyl groups, whereas in some PGA nucleophiles, which are serine residues at the N-terminus of the β chain, are β- Acts as a receptor for various other groups such as lactams and beta amino acids. PGA is also shown in Scheme 2:
Cleave a model substrate similar to penicillin G, eg, 6-nitro-3- (phenylacetamido) benzoic acid (NIPAB) of structural formula (IV) is converted to structural formula (III) Can also be characterized by the ability to cleave to phenylacetic acid and 5-amino-2-nitro-benzoic acid of structural formula (V) (eg Alkema et al., Anal. Biochem. 275: 47-53, [ 1999]). Since 5-amino-2-nitro-benzoic acid is chromogenic, the substrate of formula (IV) provides a convenient way to measure PGA activity. In addition to the reactions described above, PGA can also be used for kinetic degradation of DL-tert leucine to prepare optically pure tert leucine (see, eg, Liu et al., Prep. Biochem. Biotechnol. 36: 235-41, [2006]).

  The PGA of the present disclosure is based on an enzyme obtained from the organism Kluyvera citrophila (K. citrophila). Similar to PGA from other organisms, K.I. Citrophila PGA is a heterodimeric enzyme composed of α-subunits and β-subunits generated by proteolytic processing of pre-pro-PGA polypeptides. Removal of the signal peptide and spacer peptide produces a mature heterodimer (see, eg, Barbero et al., Gene, 49: 69-80, [1986]). K. The amino acid sequence of the naturally occurring pre-pro-PGA polypeptide of citrophila is publicly available (see, eg, GenBank Accession No. P07794, [gi: 129551]), and herein SEQ ID NO: Presented as 2. The α chain sequence of the naturally occurring K 2 citrophila PGA corresponds to residues 27-235 of SEQ ID NO: 2. The naturally occurring β-chain sequence of K.sub.3 cytophila PGA corresponds to residues 290-846 of SEQ ID NO: 2. Residues 1-26 of SEQ ID NO: 2 correspond to the signal peptide, residues 236-289 of SEQ ID NO: 2 correspond to the linking propeptide, both of which are removed to form the α chain subunit and β chain A naturally occurring mature PGA enzyme is produced, which is a heterodimer containing subunits.

  In some embodiments, the invention provides at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 relative to SEQ ID NOs: 2, 4, 6, 8, 10, and / or 12. Engineered PGA polypeptides having amino acid sequences with%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity are provided.

  The present invention provides insulin-specific deacylated biocatalysts suitable for laboratory scale preparative use. Using directed evolution, an efficient acylase variant has been developed that can completely remove the A1 / B1 / B29-triphenylacetic acid protecting group on insulin and produce> 99% free insulin. . After only 2 rounds of evolution, variants were produced that produced> 99% free insulin in less than 6 hours at an enzyme load of 0.8 g / L. The final best variant PGA_005 was improved about 8-fold over the original scaffold. The PGA variants presented herein can accept a wide range of acyl groups and exhibit increased solvent stability and improved thermostability compared to wild-type PGA. The variant PGA presented herein lacks a spacer region. Thus, no self-processing step is necessary to produce an active enzyme.

  The invention also provides polynucleotides that encode engineered PGA polypeptides. In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding the engineered PGA polypeptide can be introduced into a suitable host cell to express the corresponding PGA polypeptide.

  Due to the knowledge of codons corresponding to various amino acids, the availability of protein sequences provides an explanation of all the polynucleotides that can encode a subject. The degeneracy of the genetic code in which the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be produced that all encode the improved PGA enzyme disclosed herein. . Thus, once a particular amino acid sequence has been identified, one skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not alter the amino acid sequence of the protein. It is expected to be. In this regard, the present disclosure specifically contemplates each and every possible variation of a polynucleotide that can be made by selecting combinations based on possible codon choices, and all such variations are described in Example 5. And should be considered to be specifically disclosed for any of the polypeptides disclosed herein, including the amino acid sequences shown in the table in FIGS.

  In various embodiments, the codon is preferably selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria; preferred codons used in yeast are used for expression in yeast, and preferred codons used in mammals are: Used for expression in mammalian cells.

  In certain embodiments, the codon usage of a PGA polypeptide is optimized because the native sequence includes preferred codons and because preferred codon usage may not be required for all amino acid residues. In addition, not all codons need to be replaced. Thus, codon optimized polynucleotides encoding PGA enzymes contain preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% codon positions of the full length coding region. Can do.

  In some embodiments, the polynucleotide is at least about 85%, 86%, 87%, relative to the α and / or β chain of any of the reference engineered PGA polypeptides described herein. PGA having an amino acid sequence with sequence identity of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more It includes a nucleotide sequence that encodes a polypeptide. Thus, in some embodiments, the polynucleotide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 relative to the reference α and β chain sequences based on SEQ ID NO: 2. It encodes amino acid sequences that are%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In some embodiments, the polynucleotide encodes the α chain amino acid sequence and / or the β chain amino acid sequence of SEQ ID NO: 2.

  In some embodiments, the polynucleotide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% relative to SEQ ID NOs: 4, 6, 8, 10, and / or 12. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more nucleotide sequence encoding a PGA polypeptide having an amino acid sequence with sequence identity. Thus, in some embodiments, the polynucleotide is at least about 85%, 86%, 87%, 88%, 89%, 90 relative to SEQ ID NO: 1, 3, 5, 7, 9, and / or 11. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequences that are identical are encoded.

  In some embodiments, an isolated polynucleotide encoding an improved PGA polypeptide has been engineered in various ways to provide improved activity and / or expression of the polypeptide. . Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotide and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

  For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to create variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (eg, US Pat. Nos. 5,605,793, 5,830,721, 6th, all of which are incorporated herein by reference). 132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375 No. 6,287,861, No. 6,297,053, No. 6,576,467, No. 6,444,468, No. 5,8111238, No. 6,117, No. 679, No. 6,165,793, No. 6,180,406, No. 6,291,242, No. 6,995,017, No. 6,395,547, No. 6,506,602, 6,519,065, 6,506 03, 6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702 No. 6,391,552, No. 6,358,742, No. 6,482,647, No. 6,335,160, No. 6,653,072, No. 6 No. 6,355,484, No. 6,03,344, No. 6,319,713, No. 6,613,514, No. 6,455,253, No. 6,579,678. 6,586,182, 6,406,855, 6,946,296, 7, No. 34,564, No. 7,776,598, No. 5,837,458, No. 6,391,640, No. 6,309,883, No. 7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631, 6,528,311, 6,287 , 862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170, No. 7,620,500, No. 6,365,377, No. 6,358,740, No. 6,406,910, No. 6,413,745, No. 6,436, No. 675, No. 6,961,664, No. 7,430,477, No. 7,873,499, 7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986, 6,376 No. 246, No. 6,426,224, No. 6,423,542, No. 6,479,652, No. 6,319,714, No. 6,521,453, No. 6,521,453 No. 6,368,861, No. 7,421,347, No. 7,058,515, No. 7,024,312, No. 7,620,502, No. 7,853 410, 7,957,912, 7,904,249, and all related non-US counterparts; Ling et al., Anal. Biochem. 254 (2): 157-78 [1997]; Dale et al., Meth. Mol. Biol., 57: 369-74, [1996]; Smith. Ann. Rev. Genet., 19: 423-462, [1985]; Botstein et al., Science 229: 1193-1201, [1985]; Carter, Biochem. J., 237: 1 -7, [1986]; Kramer et al., Cell, 38: 879-887, [1984]; Wells et al., Gene, 34: 315-323, [1985]; Minshull et al., Curr. Op. Chem. Biol., 3: 284-290, [1999]; Christians et al., Nat. Biotechnol., 17: 259-264, [1999]; Crameri et al., Nature, 391: 288. 291, [1998]; Crameri et al., Nat. Biotechnol. 15: 436-438, [1997]; Zhang et al., Proc. Nat. Acad. Sci. USA, 94: 4504-4509. [1997]; Crameri et al., Nat. Biotechnol., 14: 315-319, [ 996]; Stemmer, Nature, 370: 389-391, [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91: 10747-10751, [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336).

  In some embodiments, a variant PGA acylase of the present invention further comprises additional sequences that do not alter the activity of the encoded enzyme. For example, in some embodiments, the variant PGA acylase is linked to an epitope tag, or another sequence useful in purification.

  In some embodiments, a variant PGA acylase polypeptide of the invention is secreted from a host cell that expresses it (eg, a yeast or filamentous fungal host cell) and linked to the signal peptide (ie, the amino terminus of the polypeptide). The encoded polypeptide is expressed as a preprotein comprising an amino acid sequence that directs the encoded polypeptide into the cell's secretory pathway.

  In some embodiments, the signal peptide is endogenous K. Citrophila PGA acylase signal peptide. In some other embodiments, other K.P. A signal peptide derived from a citrophila secreted protein is used. In some embodiments, other signal peptides are used depending on the host cell and other factors. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miol heinol cellinol protease, T.A. and a signal peptide coding region obtained from reesei cellobiohydrolase II. Signal peptide coding regions for bacterial host cells include, but are not limited to, Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactiformase, , NprS, nprM), and the signal peptide coding region obtained from the genes of Bacillus subtilis prsA. In some further embodiments, other signal peptides are used in the present invention (eg, Simonen and Palva, Microbiol. Rev., 57: 109-137, [1993], incorporated herein by reference). Year)). Further useful signal peptides for yeast host cells include those derived from the genes for Saccharomyces cerevisiae α-factor, Saccharomyces cerevisiae SUC2 invertase (eg, Taussig and Carlson, Nucl. Acids Res., 11: 1943). 54, [1983]; SwissProt accession number P00724; and Romanos et al., Yeast, 8: 423-488, [1992]). In some embodiments, variants of these signal peptides and other signal peptides are used. Indeed, the present invention is not limited to any particular signal peptide, as any suitable signal peptide known in the art is used in the present invention.

  In some embodiments, the present invention provides polynucleotides that encode the variant PGA acylase polypeptides described herein, and / or biologically active fragments thereof. In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory or control sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, an expression construct containing a heterologous polynucleotide encoding a variant PGA acylase is introduced into a suitable host cell to express the variant PGA acylase.

  It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, there are a large number of nucleotide sequences that encode the variant PGA acylase polypeptides of the invention. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acid of the invention where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that “U” in the RNA sequence corresponds to “T” in the DNA sequence. The present invention contemplates and provides each and every possible variation of a nucleic acid sequence encoding a polypeptide of the present invention that can be made by selecting combinations based on possible codon choices.

  As indicated above, the DNA sequence encoding PGA should be designed for codons with high codon usage bias (codons used more frequently in the protein coding region than other codons encoding the same amino acid). You can also. Preferred codons are one gene, a series of genes of common function or origin, codon usage in highly expressed genes, codon frequency in aggregated protein coding regions of the entire organism, aggregated protein coding regions of related organisms Can be determined with respect to the frequency of codon occurrence, or a combination thereof. Codons whose frequency increases with gene expression level are generally the optimal codons for expression. In particular, the DNA sequence can be optimized for expression in a particular host organism. Various to determine codon frequency (eg, codon usage, relative synonymous codon usage) and codon preference in a particular organism, including multivariate analysis (eg, using cluster analysis or correspondence analysis) As well as various methods for determining the effective number of codons used in a gene are well known in the art. A data source for obtaining codon usage can rely on any available nucleotide sequence that can encode a protein. These data sets are, as is well known in the art, nucleic acid sequences that are actually known to encode expressed proteins (eg, complete protein coding sequence-CDS), expressed sequence tags (EST), or Contains the predicted coding region of the genomic sequence. A polynucleotide encoding a variant PGA can be prepared using any suitable method known in the art. In general, oligonucleotides are synthesized individually and then combined (eg, by enzymatic or chemical ligation, or polymerase-mediated methods) to form essentially any desired contiguous sequence. In some embodiments, the polynucleotides of the invention are prepared by chemical synthesis using any suitable method known in the art, including but not limited to automated synthetic methods. For example, in the phosphoramidite method, oligonucleotides are synthesized (eg, with an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector. In some embodiments, double-stranded DNA fragments are then synthesized by synthesizing complementary strands and annealing the strands together under appropriate conditions, or using a DNA polymerase with an appropriate primer sequence. Or by adding a complementary strand. Many common standard texts that provide methods useful in the present invention are well known to those skilled in the art.

  Engineered PGAs can be obtained by subjecting a polynucleotide encoding a naturally occurring PGA to the mutagenesis and / or directed evolution methods described above. Mutagenesis can be performed according to any of the techniques known in the art, including random mutagenesis and site-directed mutagenesis. Directed evolution methods can be performed using any of the techniques known in the art for screening for improved variants, including shuffling. Other directed evolution procedures used include, but are not limited to, staggered extension process (StEP), in vitro recombination, mutagenesis PCR, cassette mutagenesis, splicing by overlap extension (SOEing), These include ProSAR ™ directed evolution methods, as well as any other suitable method.

  Clones obtained according to the mutagenesis treatment are screened for engineered PGA with the desired improved enzyme properties. Measurement of enzyme activity from the expression library can be performed using standard biochemical techniques to monitor the rate at which product is formed. If the desired improved enzyme property is thermostable, the enzyme activity can be measured after subjecting the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after heat treatment. A clone containing the polynucleotide encoding PGA is then isolated and sequenced to identify nucleotide sequence changes (if any), which are used to express the enzyme in host cells.

  Where the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases are synthesized individually and then ligated (eg, by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired sequence. A typical sequence can be formed. For example, the polynucleotides and oligonucleotides of the present invention are chemically synthesized (generally performed by automated synthetic methods, eg, Beaucage et al., Tet. Lett., 22: 1859-69, [1981 Or by using the method described in Matthes et al., EMBO J., 3: 801-05, [1984]). According to the phosphoramidite method, oligonucleotides are synthesized (eg, with an automated DNA synthesizer), purified, annealed, ligated and cloned into an appropriate vector. Furthermore, essentially any nucleic acid can be obtained from any of a variety of commercial sources (eg, The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGenh Inc.). , IL, Operon Technologies Inc., Alameda, CA, and many others).

  The present invention also provides a recombinant construct comprising a sequence encoding at least one variant PGA, as presented herein. In some embodiments, the present invention provides an expression vector comprising a modified PGA polynucleotide operably linked to a heterologous promoter. In some embodiments, the expression vectors of the invention are used to transform a suitable host cell to allow the host cell to express the variant PGA protein. Methods for recombinant expression of proteins in fungi and other organisms are well known in the art and any number of expression vectors are available or can be constructed using routine methods. In some embodiments, the nucleic acid construct of the present invention is a vector, such as a plasmid, cosmid, phage, virus, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), into which the nucleic acid sequence of the present invention is inserted. including. In some embodiments, the polynucleotides of the invention are incorporated into any one of a variety of expression vectors suitable for expressing the variant PGA polypeptide (s). Suitable vectors include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences (eg, derivatives of SV40), and bacterial plasmids, phage DNA, baculovirus, yeast plasmids, combinations of plasmid and phage DNA. Examples include viral DNA such as vectors derived from, vaccinia, adenovirus, fowlpox virus, pseudorabies, adenovirus, adeno-associated virus, retrovirus, and many others. Any suitable vector is used in the present invention that transduces the genetic material into the cell and is replicable and viable in the relevant host if replication is desired. In some embodiments, the construct further comprises a regulatory sequence, including but not limited to a promoter, operably linked to the protein coding sequence. Many suitable vectors and promoters are known to those skilled in the art. Indeed, in some embodiments, it is often useful to express a variant PGA of the invention under the control of a heterologous promoter in order to obtain a high level of expression in a particular host. In some embodiments, the promoter sequence is operably linked to the 5 'region of the variant PGA coding sequence using any suitable method known in the art. Examples of promoters useful for expressing the modified PGA include, but are not limited to, fungal promoters. In some embodiments, promoter sequences that drive expression of genes other than PGA genes in fungal strains are used. As a non-limiting example, a fungal promoter derived from a gene encoding endoglucanase can be used. In some embodiments, promoter sequences are used that drive expression of the PGA gene in fungal strains other than the fungal strain from which the PGA is derived. Examples of other suitable promoters useful for directing transcription of the nucleotide constructs of the present invention in filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase , Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehhei lipase, Aspergillus oryzae alkaline spoilase, Aspergillus oryzae alkaline spoilase Promoters derived from the genes for acetamidase and Fusarium oxysporum trypsin-like protease (see, for example, WO 96/00787, incorporated herein by reference), as well as the NA2-tpi promoter (the gene for Aspergillus niger neutral α-amylase Promoters derived from the Aspergillus oryzae triose phosphate isomerase gene, promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (for example, all herein by reference) Nunberg et al., Mol. Cell Biol. 4: 2306-2315 [1984]; Boel et al., EMBO J. Volume 3: 1581-85, pp, [1984]; and European see Patent Application No. 137,280), as well as variants thereof promoter, truncated promoter, and hybrid promoters thereof.

  In yeast host cells, useful promoters include, but are not limited to, Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae galactokinase (gal1), Saccharomyces cerevisiae alcohol dehydrogenase 3-glyceraldehyde 2-H2 GAP), and S.P. cerevisiae 3-phosphoglycerate kinase gene-derived promoter. Additional useful promoters useful for yeast host cells are known in the art (see, eg, Romanos et al., Yeast, 8: 423-488, [1992], incorporated herein by reference. See In addition, promoters associated with chitinase production in fungi are used in the present invention (eg, Blaiseau and Lafay, Gene, 120243-248, [1992], both of which are incorporated herein by reference; and Limon et al. Curr. Genet., 28: 478-83, [1995]).

  For bacterial host cells, suitable promoters for directing transcription of the disclosed nucleic acid constructs include, but are not limited to, the E. coli lac operon, the E. coli trp operon, the bacteriophage λ, the Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltose gene amylase gene (amyM), Bacillus amyliquamequylase promoters derived from the llus subtilis xylA and xylB genes and the prokaryotic β-lactamase gene (eg, Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA, 75: 3727-3731, [1978] As well as the tac promoter (see, for example, DeBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25, [1983]).

  In some embodiments, a cloned variant PGA of the present invention also has an appropriate transcription terminator sequence (a sequence that is recognized by the host cell to terminate transcription). The terminator sequence is operably linked to the 3 'end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell is used in the present invention. Exemplary transcription terminators for filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, (See also US Pat. No. 7,399,627, incorporated herein by reference). In some embodiments, exemplary terminators for yeast host cells include Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase-3-phosphate dehydrogenase Can be mentioned. Other useful terminators for yeast host cells are well known to those of skill in the art (see, eg, Romanos et al., Yeast, 8: 423-88, [1992]).

  In some embodiments, the portion of the cloned variant PGA sequence is an appropriate leader sequence (an untranslated region of the mRNA important for translation by the host cell). The leader sequence is operably linked to the 5 'end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the selected host cell is used in the present invention. Exemplary leaders for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae aldehyde Examples thereof include those obtained from the gene for 3-phosphate dehydrogenase (ADH2 / GAP).

  In some embodiments, a sequence of the invention is a polyadenylation sequence (a sequence operably linked to the 3 ′ end of a nucleic acid sequence, which is transcribed as a signal that adds a polyadenosine residue to the transcribed mRNA. Sequences that are sometimes recognized by host cells). Any polyadenylation sequence that is functional in the selected host cell is used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like glycosylase, Can be obtained. Useful polyadenylation sequences for yeast host cells are known in the art (see, eg, Guo and Sherman, Mol. Cell. Biol., 15: 5983-5990, [1995]. ).

  In some embodiments, the control sequence includes a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide, directing the encoded polypeptide into the cell's secretory pathway. The 5 'end of the coding sequence of the nucleic acid sequence may inherently contain a segment of the coding region that encodes the secreted polypeptide and a signal peptide coding region that is naturally linked within the translation reading frame. Alternatively, the 5 'end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region may be required if the coding sequence does not naturally contain a signal peptide coding region.

  Alternatively, the native signal peptide coding region can simply be replaced with an exogenous signal peptide coding region to enhance polypeptide secretion. However, any signal peptide coding region that induces a polypeptide expressed in the secretory pathway of the selected host cell can be used in the present invention.

  Effective signal peptide coding regions for bacterial host cells include, but are not limited to, Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase β-lactamase, , NprM), and the signal peptide coding region obtained from the gene of Bacillus subtilis prsA. Additional signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 57: 109-137, [1993]).

  Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miol heinol cellinol hyalinol cellinol cellulin proserum H An example is a signal peptide coding region obtained from a gene.

  Useful signal peptides for yeast host cells include, but are not limited to, the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are known in the art (see, eg, Romanos et al. [1992], supra).

  In some embodiments, the control sequence includes a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or zymogen in some cases). Propolypeptides are generally inactive and can be converted to mature active PGA polypeptides by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region is derived from Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic acid proteinase 33836).

  When both the signal peptide and the propeptide region are present at the amino terminus of the polypeptide, the propeptide region is located next to the amino terminus of the polypeptide and the signal peptide region is located next to the amino terminus of the propeptide region. Is done.

  In some embodiments, regulatory sequences are also used that allow for regulation of polypeptide expression in relation to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on and off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, by way of example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

  Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein gene amplified with heavy metals. In these cases, the nucleic acid sequence encoding the PGA polypeptide of the present invention is operably linked to regulatory sequences.

  Accordingly, in a further embodiment, the present invention provides a polynucleotide encoding an engineered PGA polypeptide or variant thereof, one or more expression control regions such as a promoter and terminator, an origin of replication, and the like. Recombinant expression vectors are provided according to the type of host to be introduced. In some embodiments, the various nucleic acids and control sequences described above are linked together to form one or more convenient restriction sites (such as insertion of a nucleic acid sequence encoding a polypeptide or Recombinant expression vectors can be produced that can contain substitutions). Alternatively, in some embodiments, the nucleic acid sequence is expressed by inserting the nucleic acid sequence, or a nucleic acid construct comprising the sequence, into an appropriate vector for expression. In creating the expression vector, the coding sequence is placed in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

  Recombinant expression vectors include any suitable vector (eg, a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can result in expression of a polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is introduced. In some embodiments, the vector is a linear plasmid or a closed circular plasmid.

  In some embodiments, the expression vector is an autonomously replicating vector (ie, a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal factor, minichromosome, or artificial chromosome. ). In some embodiments, the vector contains some means for assuring self-replication. Alternatively, in some other embodiments, the vector is integrated into the genome when introduced into a host cell and replicated along with the chromosome into which it was integrated. Furthermore, in a further embodiment, one vector or plasmid or two or more vectors or plasmids are used which together contain the total DNA introduced into the genome of the host cell or into the transposon.

  In some embodiments, the expression vectors of the invention contain one or more selectable markers that allow easy selection of transformed cells. A “selectable marker” is a gene whose product confers biocide resistance or virus resistance, resistance to antibacterial drugs or heavy metals, prototrophy to auxotrophs, and the like. Without limitation, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate) Any suitable selectable marker for use in filamentous fungal host cells is used in the present invention, including decarboxylase), sC (sulfate adenyl transferase), and trpC (anthranilate synthase), and equivalents thereof. . Additional markers useful in host cells such as Aspergillus include, but are not limited to, the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes, and the Streptomyces hygroscopicus bar gene. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Examples of bacterial selectable markers include, but are not limited to, a dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, and / or tetracycline resistance.

  In some embodiments, the expression vectors of the invention contain element (s) that allow integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the genome of a host cell, the vector is a nucleic acid sequence that encodes a polypeptide, or any of the vectors for integrating the vector into the genome by homologous or non-homologous recombination. Rely on other elements.

  In some alternative embodiments, the expression vector contains additional nucleic acid sequences that direct integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequence allows the vector to be integrated into the genome of the host cell at the exact location (s) of the chromosome (s). In order to increase the likelihood of integration in the correct position, the integration element will contain nucleotides that are highly homologous to the corresponding target sequence to enhance the probability of homologous recombination, preferably 100-10,000 base pairs. , Preferably 400-10,000 base pairs, and most preferably 800-10,000 base pairs. The integration element can be any sequence homologous to the target sequence in the genome of the host cell. Furthermore, the integration element can be a non-coding nucleic acid sequence or a coding nucleic acid sequence. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

  For autonomous replication, the vector may further include an origin of replication that allows the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origin of replication of P15A ori or plasmids pBR322, pUC19, pACYCl77 (plasmid with P15A ori), or pACYC184 that allows replication in E. coli, and pUB110, pE194, pTA1060 that allow replication in Bacillus. Or pAMβ1. Examples of origins of replication for use in yeast host cells are 2 micron origins of replication, ARS1, ARS4, ARS1 and CEN3 combinations, and ARS4 and CEN6 combinations. The origin of replication can have a mutation that renders it functional temperature sensitive in the host cell (see, eg, Ehrlich, Proc. Natl. Acad. Sci. USA, 75: 1433, [1978]). .

  In some embodiments, more than one copy of the nucleic acid sequence of the invention is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including an amplifiable selectable marker gene with the nucleic acid sequence, In this case, by culturing the cells in the presence of an appropriate selectable agent, one can select cells that contain an amplified copy of the selectable marker gene and thereby a further copy of the nucleic acid sequence.

  Many expression vectors are commercially available for use in the present invention. Suitable commercially available expression vectors include, but are not limited to, a CMV promoter, an hGH polyadenylation site for expression in mammalian host cells, a pBR322 origin of replication, and an ampicillin resistance marker for amplification in E. coli. p3xFLAGTM ™ expression vector (Sigma-Aldrich Chemicals). Other suitable expression vectors include, but are not limited to, pBluescriptII SK (-) and pBK-CMV (Stratagene), and pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or P Plasmids (see, for example, Lathe et al., Gene, 57: 193-201, [1987]).

  Thus, in some embodiments, a vector comprising a sequence encoding at least one variant PGA is transformed into a host cell to allow for propagation of the vector and expression of the variant PGA (s). In some embodiments, the variant PGA can be post-translationally modified to remove the signal peptide and, in some cases, cleaved after secretion. In some embodiments, the transformed host cell is cultured in a suitable nutrient medium under conditions that allow expression of the variant PGA (s). Any suitable medium useful for culturing host cells is used in the present invention, including, but not limited to, minimal medium or complex medium containing appropriate supplements. In some embodiments, host cells are grown in HTP medium. Suitable media are available from a variety of commercial suppliers or can be prepared according to published recipes (eg, in the catalog of the American Type Culture Collection).

  In another aspect, the invention is a polynucleotide encoding the improved PGA polypeptide presented herein, operable to one or more regulatory sequences for expressing a PGA enzyme in a host cell. A host cell comprising a polynucleotide linked to is provided. Host cells used for the expression of PGA polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli cells, Bacillus megaterium cells, Lactobacillus kefire cells, Streptomyces. Cells and Salmonella typhimurium cells; fungal cells such as yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 2011178)); insect cells such as Drosophila S2 cells and SpodopterC cells; , COS cells, BHK cells, 293 cells, and Bowes melanoma Cells; and plant cells. Suitable culture media and growth conditions for the above host cells are well known in the art.

  Polynucleotides for expressing PGA can be introduced into cells by various methods known in the art. Technologies include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. Various methods for introducing polynucleotides into cells are known to those of skill in the art.

  In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromyota, Zygomycota, Fungi impacti. In some embodiments, the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal host cells of the present invention include all filamentous forms of the subgenus Emycotina and Omycota. Filamentous fungi are characterized by vegetative mycelium with cell walls composed of chitin, cellulose and other complex polysaccharides. The filamentous fungal host cell of the present invention is morphologically distinct from yeast.

  In some embodiments of the present invention, filamentous fungal host cells include, but are not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Celipospiris, Cephalosporum, Chrysosporium, Chrysosporum, Chrysosporium, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podspora, Phlebia, Pi omyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and / or Volvariella, and / or their full generation, or incomplete generations, and the things synonymy Of any suitable genus and species, including basionyms, or taxonomic equivalents.

  In some embodiments of the invention, the host cell is a yeast cell, including but not limited to cells of the Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, yeast cells, Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia openiae, Pichia thermotolerans, Pichia sali Taria, a Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica,.

  In some embodiments of the invention, the host cell is an algal cell, such as Chlamydomonas (eg, C. reinhardtii) and Phordium (P. sp. ATCC 29409).

  In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, gram positive, gram negative and gram-variable bacterial cells. But are not limited to, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisell , Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus , S reptomyces use, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, in any suitable bacterial organisms present invention containing Yersinia and Zymomonas Is done. In some embodiments, the host cell, Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella , Streptococcus, Streptomyces, or Zymomonas species. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments, the bacterial host strain is an industrial strain. A number of bacterial industrial strains are known and suitable in the present invention. In some embodiments of the invention, the bacterial host cell is an Agrobacterium species (eg, A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarbaglutamicus, A. myorens, A. nicotianae, A. paraaffinus, A. protophonniae, A. roseparqfinus, A. sulfures, and A. urefaciens). In some embodiments of the present invention, the bacterial host cell is a Bacillus species (eg, B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. pumilus). Lautus, B. coagulans, B. brevis, B. farmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefacien). In some embodiments, host cells include, but are not limited to B.I. subtilis, B.M. pumilus, B. et al. licheniformis, B.I. megaterium, B.M. clausii, B.I. stearothermophilus, or B.I. An industrial Bacillus strain containing amyloliquefaciens. In some embodiments, the Bacillus host cell is subtilis, B.M. licheniformis, B.I. megaterium, B.M. stearothermophilus, and / or B.I. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (eg, C. acetobutylicum, C. tetani E88, C. literoseburense, C. saccharobutyricum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (eg, C. glutamicum and C. acetoacidophilum). In some embodiments, the bacterial host cell is an Escherichia species (eg, E. coli). In some embodiments, the bacterial host cell is an Erwinia species (eg, E. uredovora, E. carotovora, E. ananas, E. herbicola, E. puncata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (eg, P. citrea, and P. agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (eg, P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (eg, S. equisimiles, S. pyogenes, and S. ubelis). In some embodiments, the bacterial host cell is a Streptomyces sp. S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (eg, Z. mobilis, and Z. lipolytica).

  An exemplary host cell is E. coli W3110. An expression vector was generated by operably linking a polynucleotide encoding the improved PGA to plasmid pCK110900 operably linked to the lac promoter under the control of the lacI repressor. The expression vector also contained a P15a origin of replication and a chloramphenicol resistance gene. Cells containing the polynucleotide of interest in E. coli W3110 were isolated by subjecting the cells to chloramphenicol selection.

  A number of prokaryotic and eukaryotic strains used in the present invention are the American Type Culture Collection (ATCC), Deutsche Sulturer ul Sulchul von Mulorganismen und Zellkulturn GmbH (DSM), Central Publicly and readily available from a number of culture collections such as Collection, Northern Regional Research Center (NRRL).

  In some embodiments, the host cell is genetically modified to have properties that improve protein secretion, protein stability, and / or other properties desirable for protein expression and / or secretion. Genetic modification can be accomplished by genetic engineering techniques and / or classical microbiological techniques (eg, chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, host cells are produced using a combination of recombinant modification and classical selection techniques. Using recombinant technology, the nucleic acid molecule is introduced, deleted, inhibited or inhibited in a manner that results in increased yield of PGA variant (s) in the host cell and / or in the culture medium. Can be modified. For example, knockout of Alp1 function results in cells that are deficient in protease, and knockout of pyr5 function results in cells that have a pyrimidine deficient phenotype. In one genetic engineering approach, homologous recombination is used to induce targeted genetic modification by specifically targeting a gene in vivo in order to suppress expression of the encoded protein. Alternative approaches use siRNA, antisense and / or ribozyme technology to inhibit gene expression. Various methods for reducing the expression of a protein in a cell include, but are not limited to, deletion of all or part of the gene encoding the protein and site-directed mutagenesis that disrupts the expression or activity of the gene product. Known in the art (eg, Chaveroche et al., Nucl. Acids Res., 28:22, e97, [2000]; all incorporated herein by reference; Cho et al., Molec. Plant Microbe Interact., 19: 7-15, [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30: 1811-1817, [2008]; Takahashi et al., Mol. Gen. Genom., 272 : 344-352, [2004]; and You et al., Arch. Micriobiol., 191: 615-622, [2009]). Random mutagenesis followed by screening for the desired mutation is also used (eg Combier et al., FEMS Microbiol. Lett., 220: 141-8, [2003], both of which are incorporated by reference; and Firon Et al., Eukary. Cell, 2: 247-55, [2003]).

  Introduction of a vector or DNA construct into a host cell includes, but is not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, PEG mediated transformation, electroporation, or other common techniques known in the art. This can be accomplished using any suitable method known in the art.

  In some embodiments, engineered host cells of the invention (ie, “recombinant host cells”) are used to activate promoters, select transformants, or amplify PGA polynucleotides. And cultured in a conventional nutrient medium modified as necessary. Culture conditions such as temperature, pH, etc. are those previously used for the host cell selected for expression and are well known to those skilled in the art. As described, many standard references and texts are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archaeal origin.

  In some embodiments, cells expressing a variant PGA polypeptide of the invention are grown under batch or continuous fermentation conditions. The classic “batch fermentation” is a closed system where the medium composition is set at the start of the fermentation and is not subject to artificial replacement during the fermentation. “Fed-batch fermentation” is a variation of a batch system and is also used in the present invention. In this variation, the substrate is gradually increased and added as the fermentation progresses. The fed-batch system is useful when cell metabolism may be inhibited by catabolite repression and when it is desirable to have a limited amount of substrate in the medium. Batch fermentation and fed-batch fermentation are common and well known in the art. “Continuous fermentation” is an open system in which a defined fermentation medium is continuously added to the bioreactor and an equal volume of conditioned medium is removed for processing. In continuous fermentation, the culture is generally maintained at a constant high density where the cells are primarily in log phase growth. Continuous fermentation systems attempt to maintain steady state growth conditions. Methods for adjusting nutrients and growth factors for continuous fermentation processes and techniques for maximizing the rate at which products are formed are well known in the art of industrial microbiology.

  In some embodiments of the invention, a cell-free transcription / translation system is used for production of the variant PGA (s). Several systems are commercially available and methods are well known to those skilled in the art.

  The present invention provides methods for making variant PGA polypeptides or biologically active fragments thereof. In some embodiments, the method is at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 relative to SEQ ID NO: 2. %, At least about 97%, at least about 98%, or at least about 99%) and having been transformed with a polynucleotide encoding an amino acid sequence comprising at least one mutation presented herein Providing a host cell; culturing the transformed host cell in a culture medium under conditions that allow the host cell to express the encoded variant PGA polypeptide; and optionally expressing the variant PGA. Recovering or isolating the polypeptide and / or containing the expressed variant PGA polypeptide The culture medium in which to include recovering or isolation. In some embodiments, the method optionally lyses the transformed host cell after expression of the encoded PGA polypeptide, and optionally removes the expressed variant PGA polypeptide from the cell lysate. It is further provided to recover and / or isolate. The present invention relates to a variant comprising culturing a host cell transformed with a variant PGA polypeptide under conditions suitable for production of the variant PGA polypeptide, and recovering the variant PGA polypeptide. Further provided are methods of making PGA polypeptides. In general, recovery or isolation of PGA polypeptides is accomplished using protein recovery techniques well known in the art, including those described herein, from host cell culture media, host cells, or both. It is. In some embodiments, host cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used in protein expression include, but are not limited to, freeze-thaw cycling, sonication, mechanical disruption, and / or the use of cytolytic agents and many other suitable methods well known to those skilled in the art. Can be destroyed by any convenient method.

  Engineered PGA enzymes expressed in host cells can be any one of well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. Or a plurality can be used to recover from cells and / or culture media. A suitable solution for lysis and high efficiency extraction of proteins from bacteria such as E. coli is commercially available under the trade name CelLytic B ™ (Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide is recovered / isolated and optionally purified by any of a number of methods known in the art. For example, in some embodiments, the polypeptide can be, but is not limited to, centrifugation, filtration, extraction, spray drying, evaporation, chromatography (eg, ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion) Or isolated from the nutrient medium by conventional procedures involving precipitation. In some embodiments, a protein refolding step is used in completing the configuration of the mature protein, as desired. Further, in some embodiments, high performance liquid chromatography (HPLC) is used in the final purification step. For example, in some embodiments, methods known in the art are used in the present invention (eg, Parry et al., Biochem. J., 353: 117, both of which are incorporated herein by reference). P., [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73: 1331, [2007]). Indeed, any suitable purification method known in the art may be used in the present invention.

  Chromatographic techniques for isolating PGA polypeptides include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and are known to those skilled in the art.

  In some embodiments, affinity techniques are used to isolate PGA enzymes with improved. Any antibody that specifically binds to a PGA polypeptide may be used for affinity chromatography purification. For the production of antibodies, various host animals can be immunized by injection with PGA, including but not limited to rabbits, mice, rats, and the like. The PGA polypeptide can be attached to a suitable carrier such as BSA by a side chain functional group or by a linker attached to the side chain functional group. To increase the immunological response, depending on the host species, but not limited to, Freund (complete and incomplete), inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, Various adjuvants can be used including oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

  In some embodiments, PGA variants are prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. In some embodiments, the PGA variant is prepared as a lyophilizate, in the form of a powder (eg, acetone powder), or as an enzyme solution. In some embodiments, the PGA variant is in the form of a substantially pure preparation.

  In some embodiments, the PGA polypeptide is bound to any suitable solid substrate. Solid substrates include, but are not limited to, solid phases, surfaces, and / or membranes. Solid supports include, but are not limited to, organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, and copolymers and grafts thereof. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The shape of the substrate can be in the form of beads, spheres, particles, granules, gels, membranes or faces. The surface can be planar, substantially planar, or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be formed in the form of a well, recess or other container, container, feature or location. Multiple supports can be placed on the array at various locations where robotic delivery of reagents can be processed, or at various locations where it can be processed by detection methods and / or instruments.

  In some embodiments, PGA variants are purified using immunological methods. In one approach, a conventional method is used against a variant PGA polypeptide (eg, a polypeptide comprising any of SEQ ID NO: 2, 4, 6, 8, 10, or 12 and / or their immunity). The resulting antibody (against the original fragment) is immobilized on the beads, mixed with the cell culture medium under conditions where the modified PGA binds and precipitated. A related approach uses immunochromatography.

In some embodiments, the variant PGA is expressed as a fusion protein that includes a non-enzymatic moiety. In some embodiments, the variant PGA sequence is fused to a domain that facilitates purification. As used herein, the term “domain that facilitates purification” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include, but are not limited to, metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metal, sequences that bind glutathione (eg, GST), hemagglutinin (HA) ) Tag (corresponds to an epitope derived from influenza hemagglutinin protein; see, eg, Wilson et al., Cell, 37: 767, [1984]), maltose binding protein sequence, FLAGS extension / affinity purification FLAG epitopes utilized in the system (for example, a system available from Immunex Corp) and the like. One expression vector intended for use in the compositions and methods described herein is for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. Bring. Histidine residues can be purified by IMIAC (immobilized metal ion affinity chromatography; see, for example, Porath et al., Prot. Exp. Purif., 3: 263-281 [1992]). Facilitating, the enterokinase cleavage site provides a means for separating the variant PGA polypeptide from the fusion protein. PGEX vectors (Promega) can also be used to express foreign polypeptides as fusion proteins with glutathione S transferase (GST). In general, such fusion proteins are soluble and can be adsorbed from lysed cells to ligand-agarose beads (eg, glutathione-agarose in the case of GST fusions) followed by elution in the presence of free ligand. It can be easily purified.
Experiment

  In the following representative examples, various features and embodiments of the present disclosure are illustrated, but are exemplary and not limiting.

In the following experimental disclosure, the following abbreviations apply: ppm (parts per million); M (molar); mM (molar), uM and μM (micromolar); nM (nanomolar); g (milligram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometers); sec. (Seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (number of revolutions per minute); ° C (degrees Celsius); RT (room temperature); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); TB (Terrific Broth; 12 g / L bacto-tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7. 0, 1 mM MgSO ₄ ); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropylthiogalactoside); TFA (trifluoroacetic acid); HPLC (high performance liquid chromatography); FIOPC (positive control) Improvement magnification); HTP (high throughput); LB (Luria broth); Codexis (Code is, Inc., Redwood City, CA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Millipore (Millipore, Corp., Billerica MA); Daicel (Daicel, West Chester, PA); Genetix (Genetics USA, Inc., Beaverton, OR); Molecular Devices (Molecular Devices, LLC, Sunnyvale, CA); Applied T Department of ies, Corp., Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Thermo Scientific (Thermo Scientific, Inf., MA; Botmingen / Basel, Switzerland); Corning (Corning, Inc., Palo Alto, Calif.); And Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.); Microfluidics (Microfluidics Corp, a. .

The following sequences are used in the present invention.
SEQ ID NO: 1 (PGA WT polynucleotide sequence)
SEQ ID NO: 2 (PGA WT polypeptide sequence)
SEQ ID NO: 3 (PGA variant 1 polynucleotide sequence)
SEQ ID NO: 4 (PGA variant 1 polypeptide sequence)
SEQ ID NO: 5 (PGA variant 6 polynucleotide sequence)
SEQ ID NO: 6 (PGA variant 6 polypeptide sequence)
SEQ ID NO: 7 (PGA variant 53 polynucleotide sequence)
SEQ ID NO: 8 (PGA variant 53 polypeptide sequence)
SEQ ID NO: 9 (PGA variant 261 polynucleotide sequence)
SEQ ID NO: 10 (PGA variant 261 polypeptide sequence)
SEQ ID NO: 11 (PGA variant 258 polynucleotide sequence)
SEQ ID NO: 12 (PGA variant 258 polypeptide sequence)

Example 1
E. coli expression host containing recombinant PGA gene The first PGA enzyme used to produce the variants of the present invention was obtained from Codex® Aylase Panel (Codexis). PGA panel plates contain a collection of engineered PGA polypeptides with improved properties compared to wild type Kluyvera citrophila PGA. The wild type PGA gene is a heterodimer consisting of an α subunit (23.8 KDa) and a β subunit (62.2 KDa) linked by a 54 amino acid spacer region. Due to the presence of the spacer region, a self-processing step is required to form an active protein. The wild type gene was modified to eliminate the spacer region and thus eliminate the self-processing step. Codex® Aylase Panel (Codexis) contains PGA variants that lack a spacer region (see, eg, US Patent Application Publication No. 2010 / 0143968A1). The gene encoding PGA was cloned into the expression vector pCK110900 (see FIG. 3 of US 2006/0195947) operably linked to the lac promoter under the control of the lac1 repressor. The expression vector also contains a P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid was transformed into E. coli W3110 using standard methods known in the art. Transformants were isolated by subjecting cells to chloramphenicol selection as known in the art (see, eg, US Pat. No. 8,383,346 and WO 2010/144103).

(Example 2)
Preparation of Wet Cell Pellets Containing HTP PGA E. coli cells derived from monoclonal colonies containing the gene encoding recombinant PGA were transferred to 1% glucose and 30 μg / mL chlorampheny in the wells of a 96 well shallow well microtiter plate. Inoculated into 180 μl LB containing cole. The culture was grown overnight at 30 ° C., 200 rpm, and 85% humidity. 10 μl of each cell culture was then transferred to the wells of a 96 well deep well plate containing 390 mL TB and 30 μg / mL CAM. Deep well plates were incubated for 3 hours at 30 ° C., 250 rpm, and 85% humidity (OD600, 0.6-0.8). Cell cultures were then induced with IPTG at a final concentration of 1 mM and incubated overnight under the same conditions as originally used. The cells were then pelleted using centrifugation at 4000 rpm for 10 minutes. The supernatant was discarded and the pellet was frozen at −80 ° C. before lysis.

(Example 3)
Preparation and analysis of cell lysate containing HTP PGA First, 250 μl of lysis buffer containing 20 mM Tris-HCl buffer, pH 7.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to Example 2. Added to the cell paste in each well produced as described. The cells were lysed for 2 hours at room temperature with shaking on a desktop shaker. The plate was then centrifuged for 15 minutes at 4000 rpm and 4 ° C. Clear supernatants were used in biocatalytic reactions to determine their activity level.

  The activity of the PGA variant is based on the efficiency of the variant in removing the three phenylacetate groups chemically bound to the A1 (glycine), B1 (phenylalanine), and B29 (lysine) residues of insulin. And evaluated. The HTP reaction was performed in a 96 well deep well plate. First, 0.3 ml of the reaction mixture contained 0.1 M Tris-HCl, pH 8.0, 5 g / L triple protected insulin and 25-125 μl (depending on the linear curve) HTP lysate. HTP plates were incubated for 6 or 22 hours in a Thermotron® shaker (3 mm stroke, model number AJ185, Infors, 30 ° C., 300 rpm). The reaction was quenched with 300 μl acetonitrile and mixed for 3 minutes using a desktop shaker. The plate was then centrifuged at 4000 rpm for 2 minutes and loaded onto the HPLC for analysis. The HTP samples were analyzed using an Agilent Eclipse XDB C18, 5 μm, 2.1 × 150 mm column. The flow rate was set to 0.5 ml per minute and the temperature was set to 50 ° C. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The run time was 7.2 minutes and duplicate injections were possible. The gradient was 0.2 minutes for 75% mobile phase A, 4.9 minutes for 55% mobile phase A, 5.4 minutes for 5% mobile phase A, and 5.9 minutes back to 75% mobile phase A. It was.

Example 4
Preparation and analysis of lyophilized lysates from shake flask (SF) cultures Selected HTP cultures grown as described above were plated on LB agar plates with 1% glucose and 30 μg / ml CAM, 37 Grow overnight at ° C. Single colonies from each culture were transferred to 50 ml LB with 1% glucose and 30 μg / ml CAM. The culture was grown at 30 ° C., 250 rpm for 18 hours and subcultured approximately 1:10 in 250 ml TB containing 30 μg / ml CAM to a final OD ₆₀₀ of 0.2. Cultures were grown for 135 minutes at 30 ° C., 250 rpm to an OD ₆₀₀ of 0.6-0.8 and induced with 1 mM IPTG. The culture was then grown at 30 ° C. and 250 rpm for 20 hours. The culture was centrifuged at 4000 rpm for 20 minutes. The supernatant was discarded and the pellet was resuspended in 30 ml of 50 mM sodium phosphate, pH 7.5. Cells were pelleted (4000 rpm for 20 minutes) and frozen at −80 ° C. for 120 minutes. The frozen pellet was resuspended in 30 ml of 50 mM sodium phosphate, pH 7.5 and lysed using a Microfluidizer system (Microfluidics) at 18,000 psi. The lysate was pelleted (60 minutes at 10,000 rpm), the supernatant was frozen and lyophilized to make a shake flask (SF) enzyme.

  Altering the activity of selected shake flask PGA variants in the removal of three phenylacetate groups chemically linked to the A1 (glycine), B1 (phenylalanine), and B29 (lysine) residues of insulin Evaluation was based on body efficiency. Shake flask reactions were performed in 96 well deep well plates. First, 0.3 ml of the reaction mixture contained 0.1 M Tris-HCl, pH 8.0, 5 g / L triple protected insulin, 0.1-0.8 g / L shake flask lysate. Deep well reaction plates were incubated for 22 hours or 6 hours at 30 ° C. and 300 rpm in a Thermotron® shaker (3 mm stroke, model number AJ185, Infors) (22 hours for round 1 evolution and 6 for round 2 evolution). time). The reaction was quenched with 300 μl of acetonitrile and mixed for 3 minutes on a desktop shaker. The plate was then centrifuged at 4000 rpm for 2 minutes and loaded onto the HPLC for analysis. The HTP samples were analyzed using an Agilent Eclipse XDB C18, 5 μm, 2.1 × 150 mm column. The flow rate was set to 0.6 ml per minute and the temperature was set to 50 ° C. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The run time was 18.2 minutes and duplicate injections were possible. The gradient was 0 to 1 minute with 80% mobile phase A, 12 minutes with 60% mobile phase A, 15 minutes with 5% mobile phase A, and 16 minutes back to 80% mobile phase A.

(Example 5)
Round 1 Evolutionary Skeleton Selection, Construction and Screening Codex® Aylase Panel (Codexis) was evaluated using the shake flask protocol described in Example 4 according to the HTP protocol described in the above example. . One of the variants, referred to as “variant 1” (SEQ ID NO: 4) from Codex® Aylase Panel, in 22 hours using a 0.8 g / L shake flask lysate load 54% free insulin was produced. A substrate inhibition test was performed using variant 1 (see FIG. 1). With increasing concentrations of the triple protected insulin substrate, the production of free insulin catalyzed by variant 1 was significantly reduced. At 5 hours, free insulin production dropped from 82% at 1 g / L substrate load to 2% at 10 g / L substrate load with the enzyme load fixed at 0.8 g / L. Although the present invention is not limited to any particular mechanism, these results suggest that higher concentrations of the triple protected insulin substrate cause substrate inhibition. The amount of free insulin production increased with lower substrate concentrations (see, eg, Wang et al., Biopolymer, 25: S109-S114, [1986]). Thus, one of the advantages provided by the present invention is the generation of PGA variants that overcome substrate inhibition. This variant was selected as the backbone for the first round evolution. A homology model of variant 1 was constructed using E. coli PGA as a template (E. coli PGA has 87% sequence identity to wild-type K. citrophila PGA). Triple protected insulin was docked to the active site of variant 1 and its interaction with PGA was evaluated. The 96 positions of the amino acid sequence were then selected for the first round of evolution, whereby the first layer of active site and triple protected insulin binding site (amino acid residues within 5-6 Å) And part of the second layer (amino acid residues within 6-12 Å). Two combinatorial libraries were also designed based on PGA panel screening results and analysis of consensus mutations. All HTP screens for variants obtained in the first round of evolution used the same protocol as above, with a final reaction time of 22 hours. The active mutations with the corresponding improvement in total activity compared to variant 1 are shown in Table 5.1 below. In this table, the positive control is variant 1 (SEQ ID NO: 4).
“-”: FIOPC <0.7
“+”: FIOPC = 0.7 to 1.3
“++”: FIOPC = 1.4 to 2.0
“++++”: FIOPC ≧ 2.1

  Based on these results, variants 6, 19, 14, 67, 88, and 53 were scaled up to shake flask volumes and their activity analyzed using the protocol described in the previous examples. did. The results are shown in FIG. Variant 6 produced 93% free insulin in 22 hours at 0.8 g / L enzyme load (see FIG. 2), and a better expression level was achieved compared to variant 1. Variant 53 had the same expression level as variant 1, but variant 1 produced 73% free insulin as compared to 54% free insulin produced. Based on the results, variant 6 (SEQ ID NO: 6) was selected as the starting backbone for the next round of evolution (ie round 2). This variant was also named “expression hit”. Variant 53 (SEQ ID NO: 8) was also selected as an alternative round 2 skeleton and named “activity hit”.

(Example 6)
Round 2 library construction and screening The most beneficial mutations identified from Round 1 evolution were D484N, V264A, Q547K, V56I, S750G, V56L, S619K, V28N, V618I, and T131N. Based on the analysis of the first round results, two combinatorial libraries were designed using both variant 6 and variant 53 as scaffolds. All HTP screening methods used for round 2 variants used the protocol previously described, with a final reaction time of 6 hours. Active mutations with a corresponding improvement in total activity compared to variant 6 and variant 53 are shown in Tables 6.1 and 6.2 below. Table 6.1 provides results for variants based on variant 6, and Table 6.2 provides results for variants based on variant 53.
“-”: FIOPC <0.7
“++”: FIOPC = 0.7 to 1.3
“++”: FIOPC = 1.4 to 2.0
“++++”: FIOPC ≧ 2.1
“-”: FIOPC <0.7
“++”: FIOPC = 0.7 to 1.3
“++”: FIOPC = 1.4 to 2.0
“++++”: FIOPC ≧ 2.1

  Variants 6 (SEQ ID NO: 6), 258 (SEQ ID NO: 12) and 261 (SEQ ID NO: 10) are scaled up in shake flasks and their activity is analyzed using the protocol described in the previous examples. did. The results are shown in FIG. As shown, variant 258 produced> 99% free insulin in 6 hours at <0.8 g / L shake flask lysate load, thereby completely releasing substrate inhibition, while Variant 261 produced approximately 90% free insulin in 6 hours at 0.8 g / L lysate loading.

  Thus, the present invention provides PGA variants that have an 8-fold improvement in total activity. It was also determined that the S619K substitution has a significant effect on activity and that the D484N substitution has a major effect on expression. S619K is located in the first layer of the insulin binding site and D484N is located in the second layer of the active site.

(Example 7)
DMSO resistance DMSO resistance testing was performed on the improved PGA variants (variant numbers 6, 258, and 261). The reaction was performed in the presence of 0-50% v / v DMSO according to the protocol described in Example 4. The results (see Figure 4) show that all variants tested lose activity when DMSO is added to the test reaction. For example, at 30% v / v DMSO, only 30% of free insulin was produced by variant 258.

  All publications, patents, patent applications, and other references cited in this application are individually incorporated by reference as individual individual publications, patents, patent applications, or other references are incorporated by reference for all purposes. To the same extent as shown, it is incorporated herein by reference in its entirety for all purposes.

  While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims (10)

  An engineered penicillin G acylase capable of removing A1 / B1 / B29 triphenylacetic acid protecting group from insulin to produce free insulin, wherein the engineered penicillin G acylase is SEQ ID NO: 2, 4, At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to 6, 8, 10, and / or 12 Penicillin G acylase.   The engineered penicillin G acylase of claim 1, comprising at least one mutation provided in Table 5.1, Table 6.2, and / or Table 6.3.   2. The engineered penicillin G acylase of claim 1, comprising SEQ ID NO: 4, 6, 8, 10, or 12.   2. The engineered penicillin G acylase of claim 1, encoded by a polynucleotide sequence selected from SEQ ID NOs: 3, 5, 7, 9, and 11.   A vector comprising the polynucleotide sequence of claim 4.   A host cell comprising the vector of claim 5.   A method for producing free insulin, comprising: i) providing an engineered penicillin G acylase according to claim 1 and an A1 / B1 / B29 triphenylacetic acid protecting group, and ii) Under the conditions that the A1 / B1 / B29 triphenylacetic acid protecting group is removed by the engineered penicillin G acylase and free insulin is produced, the engineered penicillin G acylase is converted to the A1 / B1 / B29 triphenylacetic acid. Exposing to insulin comprising a protecting group.   The engineered penicillin G acylase is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% or more 8. The method of claim 7, wherein more than free insulin is produced.   9. The method of claim 7 and / or 8, wherein the penicillin G acylase comprises SEQ ID NO: 4, 6, 8, 10, or 12. A composition comprising free insulin produced according to the method of any of claims 7-9.